α-Sarcoglycan (adhalin), a 50-kDa component of the dystrophin-associated complex of proteins, participates in the stabilization of the myofiber plasma membrane in the membrane cytoskeleton. ...Deficiencies of α-sarcoglycan cause a subset of childhood-onset muscular dystrophy (SCARMD) cases. However, secondary deficiencies of α-sarcoglycan are common. To begin to establish the rates of false positives (secondary deficiencies), we used immunofluorescence to screen 30 Italian dystrophin-normal muscular dystrophy patient biopsies and identified 4 patients with partial α-sarcoglycan deficiency and 2 patients with complete deficiency. The entire α-sarcoglycan gene was screened for mutations using RT-PCR and SSCP of messenger RNA isolated from muscle biopsies in each of the six patients. Aberrant SSCP conformers and novel mutations were found only in the two complete immunohistochemical deficient patients. One patient was homozygous for a R34H amino acid substitution, while the other was a compound heterozygote (R77C, D97G). These three missense mutations, with additional mutations we and others have previously described, are all localized in the extracellular domain of α-sarcoglycan, and most result in the loss or gain of a positively charged amino acid. These data have strong implications for structure/function maps of the α-sarcoglycan molecule. Our results suggest that most patients showing partial α-sarcoglycan deficiency exhibit this as a secondary consequence of genetically distinct disorders. In support of this, we show biochemical data indicating that secondary deficiency patients show decreased immunostaining with antibodies directed against α-sarcoglycan, while having nearly normal quantities of α-sarcoglycan protein on immunoblot. This data also suggests that approximately 5% of childhood-onset dystrophin-normal muscular dystrophy patients will show a primary α-sarcoglycan deficiency.
The National Institutes of Health/Department of Energy Human Genome Project has been funding directed research for only 5 years, and it is understandably difficult to cite important research advances ...directly attributable to the project. However, the project has been constructive in fostering multidisciplinary group research and an inspiring and synergistic "just do it" attitude in both political and scientific circles, domestically and abroad. This collaborative spirit has spawned large-scale genetic and physical mapping projects, with the most impressive and useful results to date being the dense genetic maps produced by the Généthon, a French organization largely supported by the French muscular dystrophy association. With the genetic and physical map reagents now becoming available, disease-gene cloning is proceeding at an increasingly rapid pace. More important than the predictable acceleration of disease-gene mapping are the unpredictable benefits: Will a dense PCR-based dinucleotide-repeat genetic map open novel alternative approaches to disease-gene isolation? Will it become possible to localize disease genes by simply analyzing unrelated, isolated probands rather than the rarer "extended family"? Proband-based "linkage-disequilibrium cloning" may become possible if adequate density, informativeness, and stability of polymorphic loci are obtained. In addition, "genome exclusion cloning" will be added to the established positional, candidate-gene, and functional-disease-gene-cloning experimental approaches. The anticipated exponential expansion of human genetic disease information over the remainder of the 10-year tenure of the Human Genome Project unveils critical yet unresolved issues for medical education and the practice of medicine.
Identification of the biochemical deficiency that causes Duchenne muscular dystrophy (DMD) has often been hailed as the beginning of the era of ‘genetic medicine’. Identification of this first ...‘positionally cloned’ gene led to subsequent identification of dystrophin deficiency in muscles, the definition of a plethora of related muscular dystrophies and multiple species of genetically and biochemically identical animal models, and the publication of thousands of papers on dystrophin, the membrane cytoskeleton of myofibers, and molecular diagnostics. The recent advent of genome-wide approaches (single nucleotide polymorphisms, and microarrays) are quickly adding new insights into the molecular pathophysiology and underlying clinical progression of the dystrophies.
The inherited diseases hyperkalemic periodic paralysis and paramyotonia congenita are caused by mutations in the adult skeletal muscle sodium channel gene. To determine if differences in the ...expression patterns of the adult and cardiac/fetal sodium channel genes could explain some clinical features of these disorders, we developed a novel mRNA quantitation strategy called quantitative multiplex fluorescent polymerase chain reaction (QMF-PCR). This assay tests the relative levels of multiple mRNA species simultaneously using automated sequenators. We show validation of this method by competitive-PCR and RNase protection. Developmental studies of sodium channel mRNAs in humans and mice by QMF-PCR showed that the adult sodium channel mRNA quickly increased, while the cardiac/fetal sodium channel mRNA slowly decreased similarly in both limb and diaphragm muscle. We find that the adult sodium channel gene expression is predominant in fetal and neonatal muscle of both humans and mice: adult isoform mRNA concentration in fetal muscle was 8.4 x 10(-6) micrograms/micrograms of total RNA; cardiac/fetal isoform mRNA was 2.0 x 10(-6) micrograms/micrograms; and actin mRNA was 3.4 x 10(-3) micrograms/micrograms. Our results suggest that differential sodium channel gene expression correlates with age of onset of disease, but not with diaphragm involvement, in patients with hyperkalemic periodic paralysis.
In this paper, we report a new gene clustering approach, non-negative independent component analysis (nICA), for microarray data analysis. Due to positive nature of molecular expressions, nICA fits ...better to the reality of corresponding putative biological processes. In conjunction with nICA model, visual statistical data analyzer (VISDA) is applied to group genes into modules in the latent variable space. The experimental results show that significant enrichment of gene annotations within clusters can be obtained
Chapter 91 - Dystrophinopathies Hoffman, Eric P.
Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease,
2015
Book Chapter
DMD gene mutations cause biochemical deficiency of the dystrophin protein in muscle, and result in the most common of the muscular dystrophies, Duchenne muscular dystrophy (DMD). The relatively high ...incidence of gene mutations in all world populations is the result of a high de novo mutation rate of the 2.3 million base pair X-linked gene (1:10,000 sperm and oocytes). Complete loss of dystrophin in muscle causes DMD in males, while partial loss of function causes milder clinical variants (Becker muscular dystrophy). Heterozygous females can sometimes show symptoms (manifesting carriers). Dystrophin is structurally related to spectrin and other rod-shaped actin binding proteins, and is a major component of the myofiber membrane cytoskeleton. It provides a structural link between the intracellular cytoskeleton and contracting myofibrils, and the extracellular connective tissue (basal lamina). The clinical presentation of DMD is slowed motor milestone acquisition in infants (1–3 years), with initial clinical evaluation often in early school age (~3–5 years). DMD shows progressive muscle weakness and wasting. Muscle tissue shows a highly dystrophic histological picture from birth onwards (myofiber degeneration/regeneration, progressive fibrofatty infiltration). Therapeutic approaches include physical therapy, and prescription of chronic glucocorticoids. Experimental therapeutics is rapidly expanding both in preclinical models and in DMD patient clinical trials.
We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without ...detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.
An important topic in computational biology is to identify transcriptional modules through sequence analysis and gene expression profiling. A transcriptional module is formed by a group of genes ...under control of one or several transcription factors (TFs) that bind to cis-regulatory elements in the promoter regions of those genes. In this paper, we develop an integrative approach, namely motif-guided sparse decomposition (mSD), to uncover transcriptional modules by combining motif information and gene expression data. The method exploits the interplay of co-expression and co-regulation to find regulated gene patterns guided by TF binding information. Specifically, a motif-guided clustering method is first developed to estimate transcription factor binding activities (TFBAs); sparse component analysis is then followed to further identify TFs’ target genes. The experimental results show that the mSD approach can successfully help uncover condition-specific transcriptional modules that may have important implications in endocrine therapy of breast cancer.
Network Component Analysis (NCA) has shown its effectiveness in regulator identification by inferring the transcription factor activity (TFA) when both microarray data and ChIP-on-chip data are ...available. However, the NCA scheme is not applicable to many biological studies due to the lack of complete ChIP-on-chip data. In this paper, we propose an integrative NCA (iNCA) approach to combine motif information, limited ChIP-on-chip data, and gene expression data for regulatory network inference. Specifically, a Bayesian framework is adopted to develop a novel strategy, namely stability analysis with topological sampling, to infer key TFAs and their downstream gene targets. The iNCA approach with stability analysis reduces the computational cost by avoiding a direct estimation of the high-dimensional distribution in a traditional Bayesian approach. Stability indices are designed to measure the goodness of the estimated TFAs and their connectivity strengths. The approach can also be used to evaluate the confidence level of different data sources, considering the inevitable inconsistency among the data sources. The iNCA approach has been applied to a time course microarray data set of muscle regeneration. The experimental results show that iNCA can effectively integrate motif information, ChIP-on-chip data and microarray data to identify key regulators and their gene targets in muscle regeneration. In particular, several identified TFAs like those of MyoD, myogenin and YY1 are well supported by biological experiments.
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